Cell culture laser photoablation

ABSTRACT

Methods and systems for preparing clonal cell populations are described. In some instances the disclosed methods comprise: a) identifying and selecting a cell based on its position on a surface or in a container, where the selection is not based on whether the cell comprises an exogenous label or an expressed reporter; b) photoablating all non-selected cells on the surface or in the container; and c) growing a clonal population of the selected cell.

CROSS-REFERENCE

This application a continuation application of U.S. Non-Provisionalapplication Ser. No. 16/803,218, filed on Feb. 27, 2020, which claimsthe benefit of U.S. Provisional Application No. 62/811,340, filed onFeb. 27, 2019, which applications are incorporated herein by reference.

BACKGROUND

Methods and systems for creating homogeneous clonal cell populationshave become increasingly important for a variety of emergingapplications including, but not limited to, expression and purificationof genetically-engineered proteins, nucleic acids, and other cellularcomponents; production of biologic drugs (biologics); and therapeuticapplications of stem cells. One approach to creating homogeneous clonalpopulations of cells is to grow cell populations in culture, and thenselectively remove or destroy “contaminant” cells (e.g., usingmicropipettes or other physical probes, optical “tweezers”, or laserphotoablation). This approach can become increasingly cumbersome andinefficient as the number of contaminant cells, or percentage ofcontaminant cells in the cell culture, to be removed or destroyedbecomes larger. A far more efficient approach can be to isolate a singlecell prior to culturing in a contamination-free environment, therebyensuring that the resulting cell culture is a homogeneous clonal cellpopulation.

Existing methods for generating clones from single cells can focusprimarily on the use of serial dilution techniques and/or microfluidicdevices to deposit a single cell in a culture plate well or othercontainer and subsequently incubating it under appropriate conditions toensure that it divides and develops into a mature clonal population. Achallenge with the former is that random deposition of a cell suspensionthat is dilute enough to ensure that, on average, each culture platewell contains only a single cell can also ensure that many of theculture plate wells will be empty (as is well known and predicted by thePoisson distribution). This can lead to a very inefficient process interms of the number of culture plate wells that must be processed andcan also require subsequent characterization of the population in eachwell to ensure that it does indeed contain a cell culture that arosefrom a single cell. Alternatively, microfluidic device-based approachesare often prone to clogging and can subject the cells to mechanicalstress that can negatively impact their viability. Thus, there is anunmet need for new methods and technologies that provide a means forfast, efficient processing of cells and culture plates to produce clonalcell populations at a commercial scale.

SUMMARY

Disclosed herein are methods comprising, for each of one or morepartitioned surfaces or containers, a) selecting a cell based on itsposition on the partitioned surface or in the container, therebyidentifying a selected cell, wherein the selecting is not based onwhether the cell comprises an exogenous label or an expressed reporter;and b) photoablating non-selected cells on the partitioned surface or inthe container, wherein at least 90% of the one or more partitionedsurfaces or containers comprise only the selected cell as a viable cellafter the photoablating is performed. In some embodiments, at least 95%of the one or more partitioned surfaces or containers comprise only theselected cell as the viable cell after the photoablating is performed.In some embodiments, the one or more partitioned surfaces or containerscomprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100partitioned surfaces or containers.

Also disclosed herein are methods comprising: photoablating all but afirst cell of two or more cells on a surface or in a container. In someembodiments, the method further comprises growing a clonal population ofthe first cell after the photoabalating is performed. In someembodiments, the first cell is selected using an imaging technique. Insome embodiments, the method further comprises selecting the first cellusing an automated image analysis process. In some embodiments, theselecting is based on a proximity of the first cell to a center of thesurface or the container, a size of the first cell, a morphology of thefirst cell, a phenotype of the first cell, a development stage of thefirst cell, one or more biomarkers, or any combination thereof. In someembodiments, the selecting is based on one or more biomarkers, and theone or more biomarkers comprise a genetically-engineered protein. Insome embodiments, the selecting is based on the one or more biomarkers,and the one or more biomarkers comprise one or more cell surfacereceptors or one or more fluorescent signals that are derived fromfluorescent probes of cellular metabolic state. In some embodiments, theselecting is based on detection of a CRISPR editing success parameter.In some embodiments, the CRISPR editing success parameter comprises aCas-dependent fluorescent moiety. In some embodiments, theCas9-dependent fluorescent moiety is a Cas-GFP construct. In someembodiments, the two or more cells comprise about 10 to about 15 cells.In some embodiments, cells are photoablated at a rate of at least 60cells per minute. In some embodiments, cells are photoablated with anefficiency of greater than 99%. In some embodiments, cells arephotoablated using light in the wavelength range of 1440 nm to 1450 nm.

Disclosed herein are methods comprising photoablating at least 80% offive or more cells in each of a plurality of culture plate wells,wherein at least 95% of the plurality of culture plate wells containonly one viable cell after the photoablating is performed. In someembodiments, at least 98% of the plurality of culture plate wellscontain only one viable cell after the photoablating is performed. Insome embodiments, the plurality of culture plate wells comprises atleast 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 culture plate wells.

Disclosed herein are methods comprising, for each of one or morepartitioned surfaces or containers: a) selecting a cell based on itsposition on the partitioned surface or in the container, therebyidentifying a selected cell, wherein the selecting is not based onwhether the cell comprises an exogenous label or an expressed reporter;and b) photoablating non-selected cells on the partitioned surface or inthe container, wherein at least 90% of the one or more partitionedsurfaces or containers comprise only the selected cell as a viable cellafter the photoablating is performed. In some embodiments, at least 95%of the one or more partitioned surfaces or containers comprise only theselected cell as the viable cell after the photoablating is performed.In some embodiments, at least 98% of the one or more partitionedsurfaces or containers comprise only the selected cell as the viablecell after the photoablating is performed. In some embodiments, at least99% of the one or more partitioned surfaces or containers comprise onlyone viable cell after the photoablating. In some embodiments, the one ormore partitioned surfaces comprise at least 5, 10, 20, 30, 40, 50, 60,70, 80, 90, or 100 partitioned surfaces. In some embodiments, each ofthe one or more partitioned surfaces comprise at least 5, 10, 20, 30,40, 50, 60, 70, 80, 90, or 100 partitions. In some embodiments, the oneor more containers comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 containers. In some embodiments, the one or more partitionedsurfaces or containers comprises one or more partitioned surfaces,wherein the selecting comprises selecting a cell based on its positionon the one or more partitioned surfaces, wherein the photoablatingcomprises photoablating non-selected cells on the one or morepartitioned surfaces, and wherein at least 90% of the one or morepartitioned surfaces comprise only the selected cell as a viable cellafter the photoablating is performed. In some embodiments, the one ormore partitioned surfaces or containers comprises one or morecontainers, wherein the selecting comprises selecting a cell based onits position in the one or more containers, wherein the photoablatingcomprises photoablating non-selected cells in the one or morecontainers, and wherein at least 90% of the one or more containerscomprise only the selected cell as a viable cell after the photoablatingis performed. In some embodiments, the photoablating comprises use of alaser. In some embodiments, the non-selected cells are photoablatedusing light in the wavelength range of 1440 nm to 1450 nm.

Also disclosed are methods comprising: photoablating all but a firstcell of two or more cells on a surface or in a container. In someembodiments, the photoablating comprises photoablating all but a firstcell of two or more cells on a surface. In some embodiments, thephotoablating comprises photoablating all but a first cell of two ormore cells in a container. In some embodiments, the method furthercomprises growing a clonal population of the first cell after thephotoabalating is performed. In some embodiments, the method furthercomprises photodetaching one or more cells of the clonal population froma growth surface. In some embodiments, the method further comprisestesting the one or more photodetached cells. In some embodiments, themethod further comprises performing an assay on the one or morephotodetached cells. In some embodiments, the method further comprisesselecting the first cell using an imaging technique. In someembodiments, the imaging technique comprises bright-field imaging,dark-field imaging, phase contrast imaging, fluorescence imaging, or anycombination thereof. In some embodiments, the method further comprisesselecting the first cell using an automated image analysis process. Insome embodiments, the selecting is based on a proximity of the firstcell to a center of the surface or the container. In some embodiments,the selecting is based on size of the first cell. In some embodiments,the selecting is based on morphology of the first cell. In someembodiments, the selecting is based on a phenotype of the first cell. Insome embodiments, the selecting is based on a development stage of thefirst cell. In some embodiments, the selecting is based on one or morebiomarkers. In some embodiments, the one or more biomarkers comprise agenetically-engineered protein. In some embodiments, thegenetically-engineered protein comprises a green fluorescent protein(GFP) domain. In some embodiments, the one or more biomarkers compriseone or more cell surface receptors. In some embodiments, the one or morecell surface receptors are labeled with fluorescently-tagged antibodiesthat bind specifically to the one or more cell surface receptors. Insome embodiments, the one or more biomarkers comprise fluorescentsignals that are derived from one or more fluorescent probes of cellularmetabolic state. In some embodiments, the selecting is based ondetection of a CRISPR editing success parameter of the first cell. Insome embodiments, the CRISPR editing success parameter comprises aCas-dependent fluorescent moiety. In some embodiments, theCas9-dependent fluorescent moiety is a Cas-GFP construct. In someembodiments, the photoablating comprises photoablating all but a firstcell of two or more cells on a surface, and wherein the surface is asurface in a culture plate well. In some embodiments, the two or morecells comprise about 10 to about 15 cells. In some embodiments, the twoor more cells consist of 10 to 15 cells. In some embodiments, thephotoablating comprises photoablating at least 80% of five or more cellson the surface or in the container. In some embodiments, at least 90% of9 of more cells on the surface or in the container are photoablated. Insome embodiments, at least 95% of 20 or more cells on the surface or inthe container are photoablated. In some embodiments, at least 99% of 99or more cells on the surface or in the container are photoablated. Insome embodiments, cells are photoablated at a rate of at least 60 cellsper minute. In some embodiments, cells are photoablated with anefficiency of greater than 99%. In some embodiments, cells arephotoablated using light in the wavelength range of 1440 nm to 1450 nm.

Disclosed herein are methods comprising photoablating at least 80% offive or more cells in a plurality of culture plate wells, wherein atleast 95% of the plurality of culture plate wells contain only oneviable cell after the photoablating is performed. In some embodiments,at least 98% of the plurality of culture plate wells contain only oneviable cell after the photoablating is performed. In some embodiments,at least 99% of the plurality of culture plate wells contain only oneviable cell after the photoablating is performed. In some embodiments,the plurality of culture plate wells comprises at least 5, 10, 20, 30,40, 50, 60, 70, 80, 90, or 100 culture plate wells. In some embodiments,the photoablating comprises use of a laser. In some embodiments, cellsare photoablated at a rate of at least 60 cells per minute. In someembodiments, cells are photoablated with an efficiency of greater than99%. In some embodiments, cells are photoablated using light in thewavelength range of 1440 nm to 1450 nm.

Disclosed herein are methods comprising: a) providing cells in each oftwo or more partitioned surfaces or containers; b) selecting a cell ineach partitioned surface or container to retain, thereby identifying aselected cell; and c) photoablating all of the cells in each partitionedsurface or container except the selected cell, wherein cells arephotoablated at a rate of at least 60 cells per second. In someembodiments, the two or more partitioned surfaces or containers compriseat least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 partitionedsurfaces or containers. In some embodiments, the two or more partitionedsurfaces comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100partitioned surfaces. In some embodiments, each of the two or morepartitioned surfaces comprise at least 5, 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 partitions. In some embodiments, the two or morecontainers comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100 containers. In some embodiments, the method further comprisesgrowing a clonal population of the selected cell in at least one of thetwo or more partitioned surfaces or containers. In some embodiments, themethod further comprises photodetaching one or more cells of the clonalpopulation grown on or in the at least one partitioned surface orcontainer. In some embodiments, the method further comprises testing theone or more photodetached cells. In some embodiments, the selectingcomprises using an automated image analysis process. In someembodiments, the selecting is based on a proximity of the selected cellto a center of the partitioned surface or the container. In someembodiments, the selecting is based on size of the selected cell. Insome embodiments, the selecting is based on morphology of the selectedcell. In some embodiments, the selecting is based on phenotype of theselected cell. In some embodiments, the selecting is based ondevelopment stage of the selected cell. In some embodiments, theselecting is based on one or more biomarkers. In some embodiments, theone or more biomarkers comprise a genetically-engineered protein. Insome embodiments, the genetically-engineered protein comprises a greenfluorescent protein (GFP) domain. In some embodiments, the one or morebiomarkers comprise one or more cell surface receptors. In someembodiments, the one or more cell surface receptors are labeled withfluorescently-tagged antibodies that bind specifically to the one ormore cell surface receptors. In some embodiments, the one or morebiomarkers comprise fluorescent signals that are derived from one ormore fluorescent probes of cellular metabolic state. In someembodiments, the selecting is based on detection of a CRISPR editingsuccess parameter. In some embodiments, the CRISPR editing successparameter comprises a Cas-dependent fluorescent moiety. In someembodiments, the Cas9-dependent fluorescent moiety is a Cas-GFPconstruct. In some embodiments, the photoablating comprises use of alaser. In some embodiments, cells are photoablated at a rate of at least60 cells per minute. In some embodiments, cells are photoablated with anefficiency of greater than 99%. In some embodiments, cells arephotoablated using light in the wavelength range of 1440 nm to 1450 nm.

Disclosed herein are systems for preparing clonal cell populations, thesystem comprising: a) an imaging system configured to image cells ineach of one or more partitioned surfaces or containers; and b) a laserthat is optically coupled to the imaging system; wherein the system isconfigured to perform any of the methods disclosed herein. In someembodiments, the system further comprises a translation stage configuredto accurately position individual cells at a laser focal point on asample plane of the imaging system so that individual cells can bephotoablated or photodetached. In some embodiments, the system isconfigured to scan the laser relative to the one or more partitionedsurfaces or containers so that individual cells can be photoablated orphotodetached. In some embodiments, the imaging system is configured toimage cells in each of one more containers, wherein the one or morecontainers are one or more culture plate wells, and wherein the systemfurther comprises an incubator for maintaining the culture plate wellsunder a specified set of growth conditions. In some embodiments, thesystem further comprises a pick-and-place robot for moving cultureplates between translation stage and the incubator. In some embodiments,the system further comprises a controller. In some embodiments, thecontroller is configured to provide manual, semi-automated, orfully-automated control of image acquisition. In some embodiments, thecontroller is configured to provide manual, semi-automated, orfully-automated control of image processing. In some embodiments, thecontroller is configured to provide manual, semi-automated, orfully-automated control of delivery of laser light to the sample planeof the imaging system. In some embodiments, the controller is configuredto provide manual, semi-automated, or fully-automated control ofpositioning of individual cells at the laser focal point.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a block diagram of a system for laser photoablation ofcells in a cell culture according to one aspect of the presentdisclosure. In some instances, the system optionally comprises anenvironmental control chamber that encompasses the other systemcomponents. In some instances, the system controller may communicatewith and control the environmental control chamber to maintain aspecified temperature, humidity, O₂ concentration, CO₂ concentration,etc.

FIG. 2 provides a block diagram of a system for laser photoablation ofcells in a cell culture according to one aspect of the presentdisclosure. In some instances, the system optionally comprises anenvironmental control chamber or module that resides on the microscopestage and makes contact with or fully- or partially-encompasses theculture plate. In some instances, the system controller may communicatewith and control the environmental control chamber or module to maintaina specified temperature, humidity, CO₂ concentration, etc.

FIG. 3 provides a block diagram for the software used to control asystem for laser photoablation of cells in a cell culture according toone aspect of the present disclosure.

FIG. 4 provides a CAD model (isometric view) of an inverted microscopeused to perform laser photoablation of cells in a cell culture accordingto one aspect of the present disclosure.

FIG. 5 provides a CAD model (side view) of an inverted microscope usedto perform laser photoablation of cells in a cell culture according toone aspect of the present disclosure.

FIG. 6 provides a CAD model of the microscope of FIGS. 4 and 5 alongwith optical elements used to couple laser light into the microscope,and a housing (removed in this view) used to control the imagingenvironment.

FIGS. 7A-B show non-limiting examples of data that illustrate the use oflaser photoablation to remove cells and create patterns in a confluentcell culture. FIG. 7A: a pattern of cells are destroyed by laserphotoablation according to a specified set of position coordinates(box). FIG. 7B: a subsequent photoablation pattern performed on the samecell culture according to an updated set of position coordinates (box).

FIGS. 8A-B show non-limiting examples of image data that illustrate theuse of laser photoablation to destroy single cells. FIG. 8A:bright-field image of a cell culture plate showing three targeted singlecells as indicated in the boxes. FIG. 8B: bright-field image of the samecell culture plate following the destruction of the targeted cells bylaser photoablation.

FIG. 9 provides a non-limiting example of laser photoablation data wherecells were selectively ablated using a raster pattern of exposure tocontinuous laser light.

FIG. 10 provides a non-limiting example of laser photoablation datawhere cells were selectively ablated using exposure to single pulses oflaser light.

DETAILED DESCRIPTION

Disclosed herein are methods and systems for preparing clonal cellpopulations for which the confidence level that the cell populationarose from a single cell is extremely high. The approach relies on thefact that the clone production process may start by using one or moresurfaces or containers, e.g., culture plate wells, that each containmultiple cells as long as one has an efficient process for killing allbut one of the cells (or in some instances, all but two or more cells tobe retained). The one or more surfaces can be a surface of a container,e.g., a surface of a culture plate well, e.g., a bottom surface of aninterior of a culture plate well. A combination of a microscope, imageprocessing software for cell identification and position tracking, atranslation stage, and a laser can be used to create a system forablating unwanted cells on a surface or in a container, e.g., cultureplate well, in which a number of cells, e.g., a relatively small numberof cells, has first been deposited from a dilute cell suspension.Starting with one or more surfaces or containers, e.g., culture platewells, that each comprise several cells can eliminate the probleminherent in trying to deposit single cells from a cell suspension thatis sufficiently dilute to ensure that, according to the Poissondistribution, on average each surface or each container, e.g., a cultureplate well, will contain only a single cell (i.e., that an excessivenumber of surfaces or containers, e.g., culture plate wells, willcontain no cells at all), and allows one to use any of a variety ofapproaches for determining which cell (or, in some instances, acombination of cells) to keep. For example, in some instances, a singlecell (or a combination of cells) may identified and retained purely onthe basis of its location on a surface or within the container, e.g.,culture plate well (e.g., its proximity to the center of the surface orcontainer, e.g., well, e.g., bottom interior surface of the well, itsproximity to the edge of the surface or container, e.g., well, e.g.,surface of a well, or its proximity to any other defined position on thesurface or within the container, e.g., well) without reference tocell-specific features. On the other hand, in some instances, a singlecell (or a combination of cells) may be identified and retained based onone or more cell-specific criteria. Examples of criteria that may beused for selecting and retaining a cell (or for selecting and destroyinga cell) include, but are not limited to, the total number of cells on asurface or in a container, e.g., well, cell phenotype, cell morphology,the presence of one or more specified biomarkers, and/or a reportermolecule status (e.g. the presence or absence of a green fluorescentprotein (GFP) signal). In some cases, both features specific to a cell,and features not specific to a cell, are used to identify and/or selecta cell for retention.

In general, the disclosed methods may comprise: (i) delivery of one ormore cells, e.g., a cell suspension, e.g., a dilute cell suspension,onto one or more surfaces and/or into one or more containers, e.g., intoone or more culture plate wells, so that a number of cells, e.g., asmall number of cells (e.g., 5 to 10 cells) are deposited on eachsurface and/or in each container, e.g., in each well, (ii) imaging ofeach surface or container, e.g., culture plate well, (iii) processing ofone or more images for each surface or container, e.g., culture platewell, to identify individual cells and determine their position on thesurface or within the container, e.g., well, (iv) applying selectioncriteria to identify which one or more cells to retain and which todestroy, (v) programming the position coordinates of one or moreunwanted cells into a targeting system which controls a translationstage and/or laser scanning system, and (vi) exposing the one or moreunwanted cells on each surface or in each container, e.g., in eachculture plate well, to laser light to destroy them using a photoablationprocess, wherein their successful destruction is recorded as part of theablation process. In some instances, the disclosed methods may compriseany subset or any combination of these individual steps. In someinstances, imaging of one or more surfaces or containers, e.g., cultureplate wells, may comprise acquiring an image of a single field-of-viewof the surface or within the container, e.g., well, wherein the singlefield-of-view comprises all or a portion of the surface or container,e.g., well. In some instances, imaging of one or more surfaces orcontainers, e.g., culture plate wells may comprise acquiring a tiledimage created by stitching together two or more images which eachcomprise a different field-of-view of the surface or within thecontainer, e.g., well. In some instances, in addition to identifying andlocating individual cells on a surface or within a container, e.g.,well, the image processing may be used to identify and locate doublets,triplets, or other aggregates of cells, which in some instances may bedesignated for ablation and in other instances, may be designated forretention. In some instances, any or all of the individual steps of thedisclosed methods may be performed in a manual, semi-automated, orfully-automated fashion, as will be recognized by those of skill in theart.

Also disclosed herein are systems designed to perform the disclosedmethods for preparing clonal cell populations. In general, the disclosedsystems may comprise: (i) a microscope or other imaging system that isconfigured for viewing of one or more cells on a surface or in acontainer, e.g., culture dish, culture plate, culture container, orother cell culture format, (ii) a focusing system capable of re-focusingthe microscope or imaging system on individual cells as necessary, (iii)a laser and objective that are capable of working in tandem to focus anddeliver laser light to a specific location on a surface in a container,e.g., culture container, (iv) a camera or other image sensor that can beused to acquire images of one or more fields-of-view in each surface orcontainer, e.g., culture plate well or culture container, (v) atranslation stage capable of fast and accurate positioning of individualcells at the focal point of the objective, (vi) one or more processors,controllers, or computers, (vii) image capture and processing softwarefor identifying cells and determining their position coordinates in eachof a series of surfaces or containers, e.g., culture plate wells orculture containers, (viii) laser ablation control software forcontrolling laser power and/or exposure time, (ix) system controlsoftware for coordinating the image capture, image processing,translation stage movement, and laser ablation steps of the process, (x)an environmental control chamber or module that maintains the cells onthe surfaces or in the containers, e.g., culture plates or culturecontainers, under a specified set of cell culture conditions, or (xi)any combination thereof. In some instances, a laser scanning system(e.g., comprising a micromirror array positioned in the optical path bywhich laser light is delivered to the sample plane of the microscope orimaging system) may be utilized instead of, or in addition to, atranslation stage for focusing laser light onto individual cells. Insome instances, the environmental chamber may encompass all or a portionof the remaining system components. In some instances, the environmentalchamber may be configured to make contact with or encompass only thesurface or container, e.g., cell culture containers being processed,e.g., it maybe sized and adapted to be mounted on the translation stage.In some instances, the system may further comprise a fluid handlingsystem for depositing a specified volume of a cell suspension onto oneor more surfaces or into each of one or more containers, e.g., cultureplate wells or other culture containers, which may subsequently betransferred to the laser ablation system. In some instances, the systemmay further comprise robotics for moving the surfaces or containers,e.g., culture plates or culture containers, back and forth between thelaser ablation system and long-term cell culturing incubators, alongwith control software that coordinates the robotic transfer of thesurfaces or containers, e.g., culture plates or containers, with theimage capture, image processing, translation stage movement, and laserablation steps of the process. In some instances, the system may furthercomprise, e.g., barcode readers for tracking surfaces or containers,e.g., culture plates or culture containers, as they are moved in and outof the laser ablation system. In some instances, the system (or thecomputer, controller, or processor components thereof) may comprise anetwork interface for transferring surface or container, e.g., cultureplate tracking data, image data, cell identification and ablation data,and/or other experimental data from the laser ablation system to alaboratory information management system (LIMS).

The disclosed methods and systems may be applied to any of a variety ofemerging applications including, but not limited to, expression andpurification of genetically-engineered proteins, nucleic acids, andother cellular components; production of biologic drugs (biologics); andtherapeutic applications of stem cells. It shall be understood thatdifferent aspects of the disclosed methods and systems as describedherein may be appreciated individually, collectively, or in combinationwith each other.

Definitions: Unless otherwise defined, all of the technical terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art in the field to which this disclosure belongs.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Any reference to “or” herein is intended toencompass “and/or” unless otherwise stated.

As used herein, the term ‘about’ a number refers to that number plus orminus 10% of that number. The term ‘about’ when used in the context of arange refers to that range minus 10% of its lowest value and plus 10% ofits greatest value.

An example of container may a culture plate or culture plate well. Asused herein, a “culture plate” may refer to any of a variety ofmicroplate-like devices comprising a substrate and well-formingcomponent that divides the substrate into separate wells (or chambers,compartments, etc.). In some instances, a culture plate may comprise amicroplate-like device that includes a lid or cover. In some instancesthe lid or cover may be removable or detachable. In some instances, aculture plate may be a petri dish. In some instances, a culture platemay comprise a substantially flat, planar substrate that has nowell-forming component.

As used herein, the terms “laser photoablation”, “photoablation”, andsimply “ablation” are used interchangeably and in a general sense toinclude various related mechanisms by which cells may be disrupted ordestroyed upon exposure to light, e.g., intense light, at variouswavelengths (ranging from ultraviolet (UV) wavelengths to infrared (IR)wavelengths) in either a pulsed or continuous wave mode.

Clonal cell culture preparation methods: The most time consuming andcostly aspect of producing a clone can be isolation and expansion of asingle cell. As noted above, existing methods for generating cell clonesfocus on isolating a single cell in a culture container (i.e., cellsingulation) using any of a variety of techniques and technologies forseparating the cell from a mixture of cells. Isolation of single cellsmay be carried out, for example, using a cell sorting device such as theWOLF cell sorter (Nanocellect Biomedical, Inc., San Diego, Calif.), thesingle cell printer (Cytena, GmbH, Freiburg, Germany), or the YamahaCell Picker (Yamaha, Inc.). There are many other examples of cellsingulation devices (reviewed by Carey, et al. (2018), “Developments inlabel-free microfluidic methods for single-cell analysis and sorting”,WIREs Nanomed Nanobiotechnol 112: e1529-17) and those technologies areall capable of isolating a single cell from other cells for culturing.However, all of these techniques passage cells through microfluidicfeatures (<100 um in at least one dimension) that can subject a cell tomechanical stress that can decrease the fitness and viability of thecell.

The methods disclosed herein differ from the cell singulation approachesdescribed in the previous paragraph in that they bypass the difficultiesof depositing a single cell on a surface in a container, e.g., well, andinstead focus on destroying unwanted cells, e.g., once they have settledon a surface or in a container, e.g., well. There are a number ofautomated processes that can deposit several cells (e.g., 5-10 cells) in100% of the targeted culture plate wells. The disclosed methods for thenefficiently removing the unwanted cells can provide for a much higherthroughput and less costly clone production process.

As noted above, the disclosed methods may comprise all or a subset ofthe following steps: (i) delivery of cells, e.g., a cell suspension,e.g., a dilute cell suspension, onto one or more surfaces or into one ormore containers, e.g., culture plate wells so that a small number ofcells (e.g., 5 to 15 cells) are deposited on each surface or in eachcontainer, e.g., well, (ii) imaging of each surface or container, e.g.,culture plate well, (iii) processing of one or more images for eachsurface or container, e.g., culture plate well to identify individualcells and determine their position within the well, (iv) applyingselection criteria to identify which one or more cells to retain andwhich to destroy, (v) programming the position coordinates of one ormore unwanted cells into a targeting system which controls a translationstage and/or laser scanning system, and (vi) exposing the one or moreunwanted cells on each surface or in each container, e.g., culture platewell to laser light to destroy them using a photoablation process,wherein their successful destruction is recorded as part of the ablationprocess. In some instances, imaging of surfaces or containers, e.g.,culture plate wells, may comprise acquiring an image of a singlefield-of-view of the surface or within the container, e.g., well,wherein the single field-of-view comprises all or a portion of thesurface or the container, e.g., well. In some instances, imaging ofsurfaces or containers, e.g., culture plate wells may comprise acquiringa tiled image created by stitching together two or images which eachcomprise a different field-of-view of the surface or within thecontainer, e.g., well. In some instances, in addition to identifying andlocating individual cells on a surface or within a container, e.g.,well, the image processing may be used to identify and locate doublets,triplets, or other aggregates of cells, which in some instances may bedesignated for ablation and in other instances, may be designated forretention. In some instances, any or all of the individual steps of thedisclosed methods may be performed in a manual, semi-automated, orfully-automated fashion, as will be recognized by those of skill in theart.

Cells: The disclosed methods and systems may be used for preparation ofclonal populations of any of a variety of cells known to those of skillin the art. In some aspects, the cells may be any adherent andnon-adherent eukaryotic cell, mammalian cell, primary or immortalizedhuman cell or cell line, primary or immortalized rodent cell or cellline, cancer cells, normal or diseased human cells derived from any of avariety of different organs or tissue types (e.g., white blood cells,red blood cells, epithelial cells, endothelial cells, neurons, glialcells, astrocytes, fibroblasts, skeletal muscle cells, smooth musclecells, gametes, or cells from the heart, lungs, brain, liver, kidney,spleen, pancreas, thymus, bladder, stomach, colon, small intestine),distinct cell subsets such as immune cells, CD8+ T cells, CD4+ T cells,CD44^(high)/CD24^(low) cancer stem cells, Lgr5/6⁺ stem cells,undifferentiated human stem cells, human stem cells that has beeninduced to differentiate, rare cells (e.g., circulating tumor cells(CTCs), circulating epithelial cells, circulating endothelial cells,circulating endometrial cells, bone marrow cells, progenitor cells, foamcells, mesenchymal cells, or trophoblasts), animal cells (e.g., mouse,rat, pig, dog, cow, or horse), plant cells, yeast cells, fungal cells,bacterial cells, algae cells, adherent or non-adherent prokaryoticcells, or any combination thereof. In some aspects, the cells may beimmune cells, e.g., T cells, cytotoxic (killer) T cells, helper T cells,alpha beta T cells, gamma delta T cells, T cell progenitors, B cells,B-cell progenitors, lymphoid stem cells, myeloid progenitor cells,lymphocytes, granulocytes, Natural Killer cells, plasma cells, memorycells, neutrophils, eosinophils, basophils, mast cells, monocytes,dendritic cells, macrophages, or any combination thereof.

As noted, in some instances the disclosed methods and systems may beused to prepare clonal populations of stem cells, e.g., embryonic stemcells, adult (tissue-specific) stem cells, mesenchymal stem cells, orinduced pluripotent stem cells. Embryonic stem cells are obtained fromthe inner cell mass of a blastocyst (a mainly hollow ball of cells that,in the human, forms three to five days after an egg cell is fertilizedby a sperm), and are typically pluripotent, i.e., they can be used togenerate any of the body's specialized cell types, but typically cannotgenerate support structures like the placenta and umbilical cord. Adultstem cells are multipotent, i.e., they can typically generate a fewdifferent cell types found in a specific tissue or organ. Mesenchymalstem cells (MSCs; also sometimes referred to as “stromal cells”) areisolated from, e.g., bone marrow or the stroma (the connective tissuethat surrounds other tissues and organs). MSCs derived from bone marrowor other tissues have been shown to be capable of making bone, cartilageand fat cells, although it is unclear if they are actual stem cells orwhat other types of cells they are capable of generating. Theircharacteristics appear to depend on what tissue they are isolated fromand how they are isolated and grown.

In some instances, the disclosed methods and systems may be used toprepare clonal populations of induced pluripotent stem cells (IPSCs), orany differentiated cell line derived therefrom. Induced pluripotent stemcells are derived from, e.g., skin or blood cells that have beenreprogrammed to regress into an embryonic-like pluripotent state, andwhich may subsequently be triggered to differentiate into any of avariety of specific cell types, e.g., beta islet cells, egg and spermprecursors, liver cells, bone precursor cells, blood cells, neurons, andthe like, for use in biomedical research and/or therapeuticapplications.

Cell deposition: The disclosed methods for preparation of clonal cellpopulations may comprise an initial step of depositing cells onto one ormore surfaces or into one or more containers, e.g., culture plate wellsor other culture containers. An objective can be to ensure that everycontainer, e.g., well, contains at least one cell and preferably no morethan several cells. In some cases, every container, e.g., culture platewell does not comprise at least one cell. Cells may be deposited usingany of a variety of liquid-dispensing or plate-handling systems, e.g.,liquid-dispensing and plate-handling robotic systems. The cellconcentration in the stock cell suspension and/or the volume of liquiddispensed onto each surface into each container, e.g., well, can beadjusted so that on average each surface (e.g., partitioned surface) orcontainer, e.g., well, comprises a desired number of cells, e.g., 5 to10 cells. In some instances, each surface or container, e.g., well maycontain, or may contain on average, at least 1 cell, at least 2 cells,at least 3 cells, at least 4 cells, at least 5 cells, at least 6 cells,at least 7 cells, at least 8 cells, at least 9 cells, at least 10 cells,at least 11 cells, at least 12 cells, at least 13 cells, at least 14cells, at least 15 cells, at least 16 cells, at least 17 cells, at least18 cells, at least 19 cells, at least 20 cells, at least 25 cells, atleast 30 cells, at least 40 cells, or at least 50 cells. In someinstances, each surface or container, e.g., well may contain, or maycontain on average, at most 50 cells, at most 40 cells, at most 30cells, at most 25 cells, at most 20 cells, at most 19 cells, at most 18cells, at most 17 cells, at most 16 cells, at most 15 cells, at most 14cells, at most 13 cells, at most 12 cells, at most 11 cells, at most 10cells, at most 9 cells, at most 8 cells, at most 7 cells, at most 6cells, at most 5 cells, at most 4 cells, at most 3 cells, at most 2cells, or at most 1 cell. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure; for example, each surface well may contain fromabout 3 cells to about 14 cells. Those of skill in the art willrecognize that the number of cells on a surface or in a container, e.g.,well, following the initial deposition may have any value within thisrange, e.g., about 7 cells on average.

Any of a variety of liquid-dispensing and/or plate-handling robotics maybe utilized for the initial deposition of cells onto surfaces or intocontainers, e.g., culture plate wells or other culture containers.Examples include, but are not limited to, the liquid-dispensing andplate-handling robotic systems available from Agilent—Velocity 11 (MenloPark, Calif.), Beckman Coulter Life Sciences (Indianapolis, Ind.),Hamilton (Reno, Nev.), Perkin Elmer (Waltham, Mass.), Tecan (BaldwinPark, Calif.), or BioTek (Winooski, Vt.). In some cases, a roboticssystem is not used to deposit cells onto a surface or into a container,e.g., a culture plate well. In some cases, a method provided hereincomprises obtaining a surface or a container, e.g., a culture platewell, comprising one or more cells, without a user performing a celldeposition step.

Cell culturing conditions: In general, the cell culturing conditionsused (growth medium, incubation temperature, humidity, O₂ concentration,CO₂ concentration, type of culture flask, dish, or plate, etc.) willvary depending on the type of cell clones being prepared. A suitablegrowth medium provides the essential nutrients (amino acids,carbohydrates, vitamins, minerals, etc.) required by the specific celltype being cultured, maintains the pH and osmotic pressure required bythe specific cell type being cultured, and may further comprise growthfactors, hormones, etc. The cells can be anchorage-dependent cells thatcan be cultured while attached to a solid or semi-solid substrate(adherent or monolayer culture). In some cases, the cells can be grownfloating in the culture medium (suspension culture). In some instances,the disclosed methods and systems may be used to prepare clonal culturesof non-adherent cells that have been allowed to settle on the bottom ofa container, e.g., a culture plate well or culture container (or on agrowth substrate contained therein).

Imaging of cell cultures: Any of a variety of imaging techniques may beutilized as part of the disclosed methods and systems for preparingclonal cell cultures. Examples include, but are not limited to,bright-field, dark-field, phase contrast, fluorescence, and two-photonfluorescence imaging. In some instances, a super-resolution imagingtechnique may be used, e.g., super-resolution fluorescence imaging,which may allow images to be captured with a higher spatial resolution(e.g., 10-200 nm resolution) than that determined by the diffractionlimit of light at the imaging wavelength. In some instances, greyscaleimages of cells deposited in culture plate wells may be acquired andused for cell identification and determination of cell positioncoordinates. In some instances, red-green-blue (RGB, or color) images ofcells deposited in culture plate wells may be acquired and used for cellidentification and determination of cell position coordinates. Examplesof suitable imaging acquisition hardware and image processing softwarewill be discussed in more detail below.

Cell detection and selection: In some instances, images may be viewedlive by a skilled operator for identification of cells and manualcontrol of a translation stage to bring each cell to be ablated intoposition at the focal point of the laser. Images can be captured andprocessed using a semi-automated or fully-automated process to performone or more of the following steps: (i) image segmentation, (ii) featureextraction, (iii) cell identification and determination of positioncoordinates, (iv) cell selection, and (v) transfer of cell positioncoordinate data for cells selected for destruction to a targeting systemthat controls the position of the translation stage and laser exposureto selectively ablate unwanted cells.

In some instances, the selection of a single cell (or a subset of cells)to retain is made randomly and all other cells within the culture platewell or culture container are destroyed. In some instances, theselection of a single cell (or a subset of cells) to retain is made onthe basis of selection criteria that are independent of traits orproperties inherent to the cells themselves (e.g., the selecting is notbased on whether the cell comprises an exogenous label or an expressedreporter). For example, in some instances, a cell is selected to beretained simply on the basis of its location on a surface within acontainer, e.g., culture plate well. In some instances, a cell (or asubset of cells) that is closest to the center of a surface or acontainer, e.g., culture plate well or culture container is selected tobe retained, and all other cells are ablated. In some instances, a cell(or a subset of cells) that is a specified distance from the center of asurface or a container, e.g., a culture plate well or culture containeris selected to be retained, and all other cells are ablated. In someinstances, a cell (or a subset of cells) that is closest to a wall of acontainer, e.g., a culture plate well or culture container is selectedto be retained, and all other cells are ablated. In some instances, celldoubles, triplets, or other aggregates of cells will be ablatedregardless of their position on a surface or within a container, e.g., aculture plate well or culture container.

In some instances, the selection of a single cell (or a subset of cells)to retain (or destroy) is made on the basis of selection criteria thatare dependent on traits or properties inherent to the cells themselves.For example, as noted above, criteria that may be used for selecting andretaining a cell (or for selecting and destroying a cell) include, butare not limited to, cell phenotype, cell morphology, cell size,development stage, the presence or absence of one or more specifiedbiomarkers, and/or a reporter molecule status (e.g. the presence orabsence of a green fluorescent protein (GFP) signal).

In some instances, the selection of a single cell (or a subset of cells)to retain (or destroy) is made on the basis of the presence or absenceof one or more biomarkers comprising cell surface receptors and ligands,e.g., G-protein coupled receptors (GPCRs), enzyme-linked receptors, ionchannel-linked receptors, membrane-based receptor tyrosine kinases,membrane glycoproteins, etc. Examples of cell surface receptors andligands that may be used as a basis for cell selection include, but arenot limited to, angiotensin receptors, CD1a-e, CD3, CD4, CD6, CD8a-b,CD19, CD20, CD22, CD33, CD52, FGF receptors, growth hormone receptor,the KCNE1 ion channel, the KCNQ1 ion channel, the ATP1G1 Mg transporter,etc. (see, for example, Várady, et al. (2013), “Cell surface membraneproteins as personalized biomarkers: where we stand and where we areheaded”, Biomarkers Med. 7(5), 803-819, for additional examples). Insome instances, the presence or absence of one or more cell surfacebiomarkers may be detected using, e.g., one or more fluorescently-taggedantibodies that bind specifically to one of the biomarkers of interest.

In some instances, the selection of a single cell (or a subset of cells)to retain (or destroy) may be made on the basis of the presence orabsence of one or more biomarkers comprising genetically-engineeredproteins, e.g., chimeric receptors or enzymes comprise a greenfluorescence protein (GFP) domain (or a domain from any variant of GFP).In some instances, the selection of a single cell (or a subset of cells)to retain (or destroy) may be made on the basis of the presence orabsence of one or more chimeric proteins comprising a GFP domain in acell line that has been engineered to express one or more GFP-containingproteins as part of a reporter system for detection of a change incellular gene expression profiles (e.g., for the detection of anincrease or decrease of the transcription and/or translation of aspecific set of one or more genes). In some instances, the selection ofa single cell (or subset of cells) to retain (or destroy) may be made onthe basis of the presence or absence of one or more chimeric proteinscomprising a GFP domain in a cell line that has been engineered toexpress one or more GFP-containing proteins as part of a reporter systemfor detection of a change in cellular gene expression profiles due to aCRISPR editing success parameter. In some instances, the CRISPR editingsuccess parameter may comprise a Cas-dependent fluorescent moiety (e.g.,a Cas9-dependent fluorescent moiety). In some instances, a deactivatedCas (dCAS) can be tagged with XFP and in combination with a guide beused to identify cells that have been edited (Ma, H. et al. (2015),“Multicolor CRISPR Labeling of Chromosomal Loci in Human Cells”, Proc.Natl. Acad. Sci. USA 112, 3002-3007).

In some instances, the selection of a single cell (or a subset of cells)to retain (or destroy) may be made on the basis of the presence orabsence of one or more biomarkers comprising fluorescent signals thatare derived from one or more fluorescent probes of cellular metabolicstate. Examples of fluorescent probes that may be used to monitorcellular metabolic state include, but are not limited to, the“BioTracker” (Sigma-Aldrich, St. Louis, Mo.) series of fluorescent dyesfor discriminating between live cells and dead cells, fluorescent probesfor intracellular calcium²⁺ concentration (e.g., Fura 2 AM, Fura Red AM,Indo-1 AM, all from ThermoFisher Scientific, Waltham, Mass.),fluorescent probes for transmembrane potentials (e.g., FluoVolt MembranePotential Dye, di-3-ANEPPDHQ, or bis-(1,3-dibutylbarbituricacid)trimethine oxonol (DiBAC₄(3)), all from ThermoFisher Scientific,Waltham, Mass.), etc.

Once one or more cells have been selected for retention using any of theapproaches described above, the remaining unwanted cells arephotoablated and the surface or container, e.g., culture plate orculture container, may be returned to a suitable incubator or cellculture chamber for growing clonal populations of the selected cells. Insome cases, the one or more cells selected for retention are notcultured following ablation of unwanted cells. In some cases, the one ormore cells selected for retention are transferred to another surface orcontainer, e.g., following photoablation of one or more unwanted cells.In some cases, the one or more cells selected for retention are analyzedfollowing photoablation of one or more unwanted cells, e.g., the one ormore cells are subjected to single cell analysis, e.g., analysis ofnucleic acids of single cell.

Photoablation methods: The disclosed methods and systems utilize laserphotoablation (or simply “ablation”) to selectively destroy one or moreunwanted cells within a small plurality of cells on a surface or withina container, e.g., culture plate well or other culture container. Asnoted above, the term “photoablation” as used herein and as applied tothe lysis and destruction of cells may refer to a variety of relatedtechniques in which cells are subjected to an intense beam of light toselectively destroy single cells or groups of cells.

The disruption of cells can occur via a variety of different laserlight-cell interaction mechanisms that are determined primarily by theirradiance within the focal volume (Zeigler and Chiu, (2009), “LaserSelection Significantly Affects Cell Viability Following Single-CellNanosurgery”, Photochem. Photobiol. 85(5): 1218-1224). The mechanismsfor optical disruption of cells may occur over a wide range oftimescales from femtosecond (fsec) to continuous wave (cw), may comprisethe use of any of a variety of lasers, and may comprise photothermalinteractions, photoablation, or plasma-induced ablation (collectivelyreferred to as “photoablation” herein). Photothermal interactionscomprise the absorption of light by cells (or tags attached to saidcells) that leads to local heating. Formally, photoablation can occurwhen absorption of a single photon by a molecule promotes an electronfrom a bonding to a nonbonding orbital, resulting in dissociation of themolecule. Photoablation may also result in a mechanical pressure waveradiating from the focal volume, a mechanism also known as cavitation.Plasma-induced ablation can be due to a multiphoton absorption processthat results in the formation of a plasma, i.e., an ionized gascomprising positive ions and free electrons within the focal volume,which can minimize excess damage in nearby cells or tissues, and whichmay also lead to the formation of a cavitation bubble. Differentmechanisms of laser-cell interaction may lead to significantly differentoutcomes for the targeted cell, e.g., to differences in cell viability.The experimental parameters that can determine which of these mechanismsdominate in cell disruption applications can be the duration of thelaser pulse and its irradiance (Zeigler and Chiu, (2009), op. cit.).

In some instances of the disclosed methods and systems, the laser usedfor photoablation of cells may produce light at a peak wavelengthranging from about 220 nm (UV light) to about 1500 nm (IR light). Insome instances, the peak wavelength of the laser light used forphotoablation may be at least 220 nm, at least 250 nm, at least 300 nm,at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, atleast 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, atleast 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, atleast 950 nm, at least 1,000 nm, at least 1,100 nm, at least 1,200 nm,at least 1,300 nm, at least 1,400 nm, or at least 1,500 nm. In someinstances, the peak wavelength of the laser light used for photoablationmay be at most 1,500 nm, at most 1,400 nm, at most 1,300 nm, at most1,200 nm, at most 1,100 nm, at most 1,000 nm, at most 950 nm, at most900 nm, at most 850 nm, at most 800 nm, at most 750 nm, at most 700 nm,at most 650 nm, at most 600 n, at most 550 nm, at most 500 nm, at most450 nm, at most 400 nm, at most 350 nm, at most 300 nm, at most 250 nm,or at most 220 nm. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the peak wavelength of thelaser light used for photoablation may range from about 1,300 nm toabout 1,500 nm. Those of skill in the art will recognize that the peakwavelength of the laser light used for photoablation may have any valuewithin this range, e.g., about 1,460 nm.

In some instances of the disclosed methods and systems, the laser usedfor photoablation of cells may produce light having a bandwidth (e.g.,full width at half maximum (FWHM)) centered on or near the peakwavelength that ranges from about 0.0001 nm to about 10 nm, depending onpeak wavelength and whether the laser is a continuous wave laser orpulsed laser. In some instances, the bandwidth may be at least 0.0001nm, at least 0.001 nm, at least 0.01 nm, at least 0.1 nm, at least 1 nm,or at least 10 nm. In some instances, the bandwidth may be at most 10nm, at most 1 nm, at most 0.1 nm, at most 0.01 nm, at most 0.001 nm, orat most 0.0001 nm. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the bandwidth may range fromabout 0.001 nm to about 1 nm. Those of skill in the art will recognizethat the bandwidth of the laser light used for photoablation may haveany value within this range, e.g., about 0.25 nm.

In some instances of the disclosed methods and systems, the laser usedfor photoablation of cells may produce continuous wave light, and anelectro-optic modulator or electronic shutter may be used to createpulses of light of arbitrarily long duration (e.g., ranging from tens ofpicoseconds to seconds). In some instances of the disclosed methods andsystems, the laser used for photoablation of cells may be a pulsedlaser, and may produce light pulses having a duration ranging from about1 femtosecond to about 100 milliseconds. In some instances, the lightpulses used for photoablation may be at least 1 femtosecond, at least 1picosecond, at least 1 nanosecond, at least 1 millisecond, at least 10milliseconds, at least 100 milliseconds, or at least 1 second induration. In some instances, the light pulses used for photoablation maybe at most 1 second, at most 100 milliseconds, at most 10 milliseconds,at most 1 millisecond, at most 1 nanosecond, at most 1 picosecond, or atmost 1 femtosecond in duration. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances the lightpulses used for photoablation may range from about 1 picosecond to about1 nanosecond in duration. Those of skill in the art will recognize thatthe pulse duration of the laser light used for photoablation may haveany value within this range, e.g., about 0.250 nanoseconds.

In some instances of the disclosed methods and systems, the laser lightused for photoablation of cells may be pulsed at a pulse repetitionfrequency ranging from about 1 Hz to about 100 MHz, depending on thetype of laser used. In instances, the pulse repetition frequency may beat least 1 Hz, at least 10 Hz, at least 100 Hz, at least 1 KHz, at least10 KHz, at least 100 KHz, at least 1 MHz, at least 10 MHz, or at least100 MHz. In some instances, the pulse repetition frequency may be atmost 100 MHz, at most 10 MHz, at most 1 MHz, at most 100 KHz, at most 10KHz, at most 1 KHz, at most 100 Hz, at most 10 Hz, or at most 1 Hz. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the pulse repetition rate may range fromabout 10 Hz to about 1 MHz. Those of skill in the art will recognizethat the pulse repetition rate may have any value within this range,e.g., about 16.5 KHz.

In some instances, the laser light irradiance (i.e., the radiant flux(power) delivered per unit area of surface, as measured, e.g., in unitsof W/cm²) may range from about 0.1 W/cm² to about 10¹⁰ W/cm², dependingon the type of laser used and the size of the focal spot at the sampleplane. In some instances, the radiant flux delivered to the samplesurface may be at least 0.1 W/cm², at least 1 W/cm², at least 10 W/cm²,at least 100 W/cm², at least 1,000 W/cm², at least 10⁴ W/cm², at least10⁵ W/cm², at least 10⁶ W/cm², at least 10⁷ W/cm², at least 10⁸ W/cm²,at least 10⁹ W/cm², or at least 10¹⁰ W/cm². In some instances, theradiant flux delivered to the sample surface may be at most at most 10¹⁰W/cm² at most 10⁹ W/cm², at most 10⁸ W/cm², at most 10⁷ W/cm², at most10⁶ W/cm², at most 10⁵ W/cm², at most 10⁴ W/cm², at most 1,000 W/cm², atmost 100 W/cm², at most 10 W/cm², at most 1 W/cm², or at most 0.1 W/cm².Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the radiant flux delivered to the samplesurface may range from about 10 W/cm² to about 1,000 W/cm². Those ofskill in the art will recognize that the radiant flux delivered to thesample surface may have any value within this range, e.g., about 0.8W/cm².

In some instances of the disclosed methods and systems, unwanted cellsmay be photoablated at a rate ranging from about 10 cells per minute toabout 200 cells per minute. In some instances, unwanted cells may bephotoablated at a rate of at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 110, at least 120, at least 130, at least140, at least 150, at least 160, at least 170, at least 180, at least190, or at least 200 cells per minute. In some instances, unwanted cellsmay be photoablated at a rate of at most 200, at most 190, at most 180,at most 170, at most 160, at most 150, at most 140, at most 130, at most120, at most 110, at most 100, at most 90, at most 80, at most 70, atmost 60, at most 50, at most 40, at most 30, at most 20, or at most 10cells per minute. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances unwanted cells may bephotoablated at a rate ranging from about 50 cells per minute to about180 cells per minute. Those of skill in the art will recognize that thephotoablation rate may have any value within this range, e.g., about 64cells per minute.

In some instances of the disclosed methods and systems, multiplesurfaces or containers, e.g., culture plate wells (or other culturecontainers (or partitions in a partitioned surface)) may be processed(i.e., all unwanted cells in the surface or container, e.g., cultureplate well or other culture container/partition, are destroyed using thedisclosed photoablation methods) at a rate ranging from about 4 wellsper minute to about 20 wells per minute. In some instances, cultureplate wells may be processed at a rate of at least 4, at least 6, atleast 8, at least 10, at least 12, at least 14, at least 16, at least18, or at least 20 wells per minute. In some instances, culture platewells may be processed at a rate of at most 20, at most 18, at most 16,at most 14, at most 12, at most 10, at most 8, at most 6, or at most 4wells per minute. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances culture plate wells may beprocessed at a rate ranging from about 6 wells per minute to about 18wells per minute. Those of skill in the art will recognize that the rateat which culture plate wells are processed may have any value withinthis range, e.g., about 15 wells per minute.

In some instances of the disclosed methods and systems, thephotoablation step may comprise ablating between about 80% and about 99%of the cells in a container, e.g., culture plate well (or other culturecontainers (or partitions in a partitioned surface)). In some instances,at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the cells on asurface or in a container, e.g., culture plate well are photoablated,where the number of cells initially on the surface contained or in thecontainer, e.g., well is at least 5, at least 10, at least 20, at least30, at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 125, at least 150, at least 175, atleast 200 cells, at least 300 cells, at least 400 cells, or at least 500cells. Any combination of ablation percentages and number of cellsinitially on a surface or contained in a container, e.g., culture platewell or other culture container described above is included in thepresent disclosure.

In some instances of the disclosed methods and systems, the efficiencyof the photoablation reaction in rendering the cells selected fordestruction as non-viable ranges from about 90% to about 99.99%, orhigher. In some instances, the efficiency of the photoablation step isat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least99.8%, at least 99.9%, or at least 99.99%. In some instances, theefficiency of the photoablation step is at most 99.99%, at most 99.9%,at most 99.8%, at most 99.7%, at most 99.6%, at most 99.5%, at most 99%,at most 98%, at most 97%, at most 96%, at most 95%, or at most 90%. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the efficiency of the photoablation step mayrange from about 95% to about 99.8%. Those of skill in the art willrecognize that the efficiency of the photoablation step may have anyvalue within this range, e.g., about 99.85%.

In some instances of the disclosed methods and systems, the efficiencyof the photoablation step is such that the percentage of containers,e.g., culture plate wells (or other culture containers or partitions ofa partitioned surface) that retain a single viable cell ranges fromabout 90% to about 99.99%, or higher. In some instances, the percentageof processed containers, e.g., culture plate wells that retain a singleviable cell is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, at least 99.9%, or at least 99.99%. In someinstances, the percentage of processed containers, e.g., culture platewells that retain a single viable cell is at most 99.99%, at most 99.9%,at most 99.8%, at most 99.7%, at most 99.6%, at most 99.5%, at most 99%,at most 98%, at most 97%, at most 96%, at most 95%, or at most 90%. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the percentage of processed culture platewells that retain a single viable cell may range from about 95% to about99.8%. Those of skill in the art will recognize that the percentage ofprocessed containers, e.g., culture plate wells that retain a singleviable cell may have any value within this range, e.g., about 99.74%.

Photodetachment methods: In some instances of the disclosed methods andsystems, the same laser used for performing photoablation, or adifferent laser, may optionally be configured for performingphotodetachment of cells from a surface on which they are growing ratherthan photoablation. Photodetachment may be used, for example, to detachand retrieve selected cells for subsequent testing (e.g., genetictesting) and/or culturing in a secondary culture plate or culturecontainer.

Laser-based photodetachment offers a non-lethal means to dissociateadherent cells from a substrate on which they are grown withoutrequiring chemical dissociation reagents. Adjustments to power settings,pulse-width modulation, and focal plane of the laser can be adjusted insuch a way to create an energy pulse that effectively detaches theselected cells without destroying the cell membranes.

Focused laser light may, for example, be scanned across a region beneathor adjacent to one or more selected cells to detach the cells from asurface on which they are growing. In some instances, illumination bythe focused laser light may result in a photothermal detachment of theone or more selected cells. In some instances, illumination by thefocused laser light may result in a photomechanical detachment of theone or more selected cells. In some instances, illumination by thefocused laser light may result in a photoacoustic detachment of the oneor more selected cells. In some instances, the cell culture plate orculture container may comprise one or more surface coating layers thathave been specially formulated to facilitate detachment of cells growingthereon by means of a photothermal and/or photomechanical detachmentmechanism. In some instances, the cell culture plate or culturecontainer may comprise one or more surface coating layers that comprisea photocleavable linker which tethers a cell recognition element, e.g.,an antibody directed towards a cell surface receptor, to a surfacewithin the cell selection compartment, where the cell recognitionelement is used to capture and tether suspension cells to a surface andwhere, upon illumination by focused laser light of the appropriatewavelength, the photocleavable linker is disrupted and a set of selectedcells may be released from the surface.

Under static conditions, cells that have been detached may settle backdown on the growth surface within the culture plate or container. Insome instances, laser-based photodetachment may thus be performed inconjunction with providing a directed flow of fluid across the growthsurface to direct the detached cells towards, e.g., a cell removal portthrough which they may be withdrawn from the cell culture device. Thecombination of laser-based photodetachment and flow-directed removal ofdetached cells allows one to remove targeted cells without riskingcontamination through manual intervention (e.g., through the use ofmedia changes or chemical dissociation reagents).

In some instances, one or more lasers may be used for performinglaser-induced photodetachment. In some instances, photodetachment may beperformed using lasers operating in the ultraviolet (UV), visible, ornear-infrared (near-IR) regions of the electromagnetic spectrum. In someinstances, laser photodetachment may be performed using laser light in awavelength range of about 1440 nm to about 1450 nm.

In some instances, one or more of the lasers used for photodetachment(or for photoablation) may be continuous wave lasers. In some instances,one or more of the lasers used for photodetachment (or forphotoablation) may be pulsed lasers. Depending on the type of laserselected and the technique used to generate pulses (e.g., mode-lockedsolid-state laser, Q-switched solid-state laser, or gain switchedsemiconductor laser), laser pulse frequencies may range from less than 1Hz to greater than 100 GHz. Similarly, depending on the type of laserselected and the technique used to generate pulses, laser pulse widthsmay range from longer than 1 microsecond to fewer than 100 femtoseconds.

In some instances, the same one or more lasers may be used to performphotodetachment and/or photoablation. In some instances, differentlasers may be used to perform photodetachment, and/or photoablation. Inthe case that the same laser or set of lasers is used to performphotodetachment and/or photoablation, the system used in conjunctionwith the disclosed methods and culture plates or containers may beoperably switched between a a photodetachment operating mode and aphotoablation operating mode by controlling laser spot size, laser spotshape, laser light intensity, laser pulse frequency, laser pulse energy,the total number of laser pulses delivered at a specified position on asurface or within the volume of the cell culture plate or culturecontainer, the position of the laser focal point relative to the surfaceor within the volume of the cell culture plate or cell culturecontainer, or any combination thereof.

In some instances, the efficiency of laser-induced photodetachment mayrange from about 50% to about 100%. In some instances, the efficiency oflaser-induced photodetachment may be at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or about 100%. In some instances, theefficiency of laser-induced photodetachment may be at most 100%, at most99%, at most 98%, at most 95%, at most 90%, at most 85%, at most 80%, atmost 70%, at most 60%, or at most 50%. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances theefficiency of laser-induced photodetachment may range from about 60% toabout 95%. Those of skill in the art will recognize that the efficiencyof laser-induced photodetachment may have any value within this range,e.g., about 93%.

Cell removal and testing: In some instances, cells that have beendetached from a growth surface within a cell culture plate or culturecontainer may optionally be removed from the cell culture device andsubjected to further testing. Examples of testing (or assays) to whichthe one or more cells removed from the cell culture plate or culturecontainer may be subjected to include, but are not limited to, nucleicacid sequencing, gene expression profiling, detection of a modified RNAmolecule, DNA molecule, or gene, detection of a CRISPR edited gene, arestriction site analysis of nucleic acid molecules, detection of aprotein (e.g., a specific biomarker protein, a mutant protein, areporter protein, or a genetically-engineered protein, and the like),detection of a change in an intracellular signaling pathway due to analtered protein function.

In some instances, the testing may be performed on a single cell thathas been detached from a clonal cell colony and removed from the cellculture device. In some instances, the number of cells (e.g., the subsetof cells that have been detached and removed from a single clonal cellcolony) that are removed from the device for each clonal cell colonyselected for testing may be fewer than 200 cells, fewer than 100 cells,fewer than 50 cells, fewer than 40 cells, fewer than 30 cells, fewerthan 20 cells, fewer than 10 cells, or fewer than 5 cells.

System & system components: Disclosed herein are systems configured toperform the methods described above. As noted above, in some instancesthe disclosed systems may comprise (i) a microscope or other imagingsystem that is configured for viewing of cells on a surface or in acontainer, e.g., culture dish, culture plate, culture container, orother cell culture format, (ii) a focusing system capable of re-focusingthe microscope or imaging system on individual cells as necessary, (iii)a laser and objective that are capable of working in tandem to focus anddeliver laser light to a specific location in a culture container, (iv)a camera or other image sensor that can be used to acquire images of oneor more fields-of-view on each surface or in each container, e.g.,culture plate well or culture container, (v) a translation stage capableof fast and accurate positioning of individual cells at the focal pointof the objective, (vi) one or more processors, controllers, orcomputers, (vii) image capture and processing software for identifyingcells and determining their position coordinates in each of a series ofcontainers, e.g., culture plate wells or culture containers, (viii)laser ablation control software for controlling laser power and/orexposure time, (ix) system control software for coordinating the imagecapture, image processing, translation stage movement, and laserablation steps of the process, (x) an environmental control chamber ormodule that maintains the cells on the surface or in the containers,e.g., culture plates or culture containers under a specified set of cellculture conditions, or (xi) any combination thereof.

In some instances, a laser scanning system (e.g., comprising amicromirror array positioned in the optical path by which laser light isdelivered to the sample plane of the microscope or imaging system) maybe utilized instead of, or in addition to, a translation stage forfocusing laser light onto individual cells. In some instances, theenvironmental chamber may encompass all or a portion of the remainingsystem components. In some instances, the environmental chamber may beconfigured to make contact with or encompass only the surfaces orcontainers, e.g., cell culture containers being processed, e.g., itmaybe sized and adapted to be mounted on the translation stage. In someinstances, the system may further comprise a fluid handling system fordepositing a specified volume of a cell suspension into each of one ormore surfaces or containers, e.g., culture plate wells or other culturecontainers, which may subsequently be transferred to the laser ablationsystem. In some instances, the system may further comprise robotics(e.g., plate-handling robotics or pick-and-place robotics) for movingsurfaces or containers, e.g., culture plates or culture containers, backand forth between the laser ablation system and long-term cell culturingincubators, along with control software that coordinates the robotictransfer of surfaces of containers, e.g., culture plates or culturecontainers with the image capture, image processing, translation stagemovement, and laser ablation steps of the process. In some instances,the system may further comprise, e.g., barcode readers for trackingsurfaces or container, e.g., culture plates or culture containers, asthey are moved in and out of the laser ablation system. In someinstances, the system (or the computer, controller, or processorcomponents thereof) may comprise a network interface for transferringcontainer, e.g., culture plate tracking data, image data, cellidentification and ablation data, and/or other experimental data fromthe laser ablation system to a laboratory information management system(LIMS).

FIG. 1 provides a schematic illustration of one non-limiting example ofa system of the present disclosure. The system may comprise a microscopeand camera (e.g., a CMOS or CCD camera) configured to capture images ofan individual well of a container, e.g., culture plate (or other culturecontainer) positioned at the focal plane for the microscope and at thefocal point of a beam of laser light introduced via the microscopeoptics. In some instances, the system may comprise an autofocus modulethat re-focuses the microscope on a surface (e.g., a growth mediumsurface) within a culture plate well or container upon repositioning ofthe culture plate or container using a translation stage. In someinstances, the control of the translation stage and positioning of thecontainer, e.g., culture plate well, capture of images, selection ofcells for ablation, and control of laser light exposure may be performedin a manual, semi-automated, or fully-automated manner. In someinstances, a controller may execute a program comprisingsoftware-encoded instructions for coordinating and controlling thepositioning of the translation stage, the capture of images, automatedprocessing of images to identify cells and select a subset of cells forablation, repositioning of the translation stage to target the selectedsubset of cells for exposure to laser light, and/or the exposure ofcells to laser light, or any combination thereof. In some instances, asillustrated in FIG. 1, the entire laser ablation system may be housedwithin an environmental control chamber that excludes room light and/orprovides control of environmental parameters such as temperature,humidity, O₂ concentration, CO₂ concentration, etc. In some instances,as illustrated in FIG. 2, the environmental control chamber mayencompass only the container, e.g., culture plate being processed, or astack of containers, e.g., culture plates ready to be processed.

Microscope or imaging system: In some instances, the disclosed systemsmay comprise a microscope equipped with a camera (or a custom imagingmodule) configured to capture images of containers, e.g., culture platewells or other culture containers (including partitions on a partitionedsurface). In some instances, the microscope may comprise acommercially-available microscope system, e.g., an upright, inverted, orepifluorescence microscope. In some instances, the microscope or imagingmodule may comprise one or more cameras or image sensors, light sources,objective lenses, additional lenses, prisms, diffraction gratings,mirrors, optical filters, colored glass filters, narrowband interferencefilters, broadband interference filters, dichroic reflectors, opticalfilters, apertures, optical fibers, optical waveguides, and the like, orany combination thereof.

In some instances, the microscope or imaging module of the disclosedsystems may comprise an autofocus mechanism that re-focuses themicroscope or imaging module on a surface (e.g., a growth medium surfaceor the bottom of a well) within a container, e.g., culture plate well,upon repositioning of the culture plate using a translation stage.

Any of a variety of light sources may be used to provide the imaging orexcitation light, including but not limited to, tungsten lamps,tungsten-halogen lamps, arc lamps, lasers, light emitting diodes (LEDs),or laser diodes. In some instances, a combination of one or more lightsources, and additional optical components, e.g. lenses, filters,apertures, diaphragms, mirrors, and the like, will comprise anillumination sub-system.

Any of a variety of image sensors may be used for imaging purposes,including but not limited to, charge-coupled device (CCD) cameras orsensors, image intensified CCD cameras or sensors, CMOS image cameras orsensors, and the like. In some instances, a combination of one or moreimage sensors, and additional optical components, e.g. lenses, filters,apertures, diaphragms, mirrors, and the like, will comprise an imagingsub-system.

Imaging mode: Any of a variety of imaging modes may be utilized inimplementing the disclosed methods. Examples include, but are notlimited to, bright-field imaging, dark-field imaging, phase contrastimaging, fluorescence imaging, super-resolution fluorescence imaging,two-photon fluorescence imaging, and the like. In some instances, dualwavelength excitation and emission (or multi-wavelength excitation oremission) fluorescence imaging may be performed.

In some instances, each surface or container, e.g., culture plate well(or culture container) may be imaged in its entirety within a singleimage, i.e., the field-of-view (FOV) may encompass the entire well,depending on the magnification used. In some embodiments, a series ofimages comprising a smaller FOV may be “tiled” or “stitched” to create ahigh-resolution image of the entire surface or container, e.g., well. Insome instances, a series of one or more images may be acquired of all ora portion of a surface or container, e.g., culture plate well. In someinstances, a series of two or more images may comprise images acquiredboth before and after performing the ablation step to destroy unwantedcells. In some instances, one or more images acquired after performingthe ablation step may be used to confirm that the selected cell(s) havebeen destroyed. In some instanced, a series of images may comprise 1, 2,3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more images.

Image processing: In some instances of the disclosed methods andsystems, image pre-processing and/or image processing may be performedin a manual, semi-automated, or fully-automated manner. In someinstances, a series of one or more images may be pre-processed to, forexample, correct image contrast and brightness, correct for non-uniformillumination, correct for an optical aberration (e.g., a sphericalaberration, a chromatic aberration, etc.), remove noise, etc., or anycombination thereof. In some instances, a series of one or more imagesmay be processed to, for example, identify objects (e.g., cells orsub-cellular structures) within each of the images, segment each of theimages to isolate the identified objects, tile segmented images tocreate composite images, perform feature extraction (e.g.,identification and/or quantitation of object properties such asobservable cellular phenotypic traits), determining the positioncoordinates for one or more selected cells, determining a confidencelevel for the destruction of the selected cells from one or more imagesacquired after performing the ablation step, or any combination thereof.

Any of a variety of image processing methods known to those of skill inthe art may be used for image processing to identify objects within theimages. Examples include, but are not limited to, Canny edge detectionmethods, Canny-Deriche edge detection methods, first-order gradient edgedetection methods (e.g., the Sobel operator), second order differentialedge detection methods, phase congruency (phase coherence) edgedetection methods, other image segmentation algorithms (e.g., intensitythresholding, intensity clustering methods, intensity histogram-basedmethods, etc.), feature and pattern recognition algorithms (e.g., thegeneralized Hough transform for detecting arbitrary shapes, the circularHough transform, etc.), image texture analysis methods (e.g., gray-levelco-occurrence matrices), and mathematical analysis algorithms (e.g.,Fourier transform, fast Fourier transform, wavelet analysis,auto-correlation, etc.), or any combination thereof.

Photoablation and/or photodetachment laser(s): Any of a variety oflasers may be used for photoablation and/or photodetachment purposes.Examples include, but are not limited to, diode (or semiconductor)lasers, solid-state lasers, gas lasers, and excimer lasers. Diode laserscan provide compact, relatively low power light sources that areavailable for a variety of wavelengths. Solid state lasers can havelasing material distributed in a solid matrix, e.g., the ruby orneodymium-YAG (yttrium aluminum garnet) lasers. The neodymium-YAG lasercan emit infrared light at 1.064 micrometers. Gas lasers, e.g., heliumand helium-neon (HeNe) lasers can have a primary output of visible redlight. CO₂ lasers can emit energy in the far-infrared (10.6 micrometers)and can be used for cutting hard materials. Excimer lasers can usereactive gases such as chlorine and fluorine mixed with inert gases suchas argon, krypton, or xenon which, when electrically stimulated producea pseudomolecule or dimer, and when lased produce light in theultraviolet wavelength range.

As noted above, the laser used for photoablation and/or photodetachmentof cells in the disclosed methods and systems may produce light at apeak wavelength ranging from about 220 nm (UV light) to about 1500 nm(IR light). In some instances, the peak wavelength of the laser lightused for photoablation and/or photodetachment may be at least 220 nm, atleast 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, atleast 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, atleast 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, atleast 850 nm, at least 900 nm, at least 950 nm, at least 1,000 nm, atleast 1,100 nm, at least 1,200 nm, at least 1,300 nm, at least 1,400 nm,or at least 1,500 nm. In some instances, the peak wavelength of thelaser light used for photoablation and/or photodetachment may be at most1,500 nm, at most 1,400 nm, at most 1,300 nm, at most 1,200 nm, at most1,100 nm, at most 1,000 nm, at most 950 nm, at most 900 nm, at most 850nm, at most 800 nm, at most 750 nm, at most 700 nm, at most 650 nm, atmost 600 n, at most 550 nm, at most 500 nm, at most 450 nm, at most 400nm, at most 350 nm, at most 300 nm, at most 250 nm, or at most 220 nm.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the peak wavelength of the laser light usedfor photoablation and/or photodetachment may range from about 1,300 nmto about 1,500 nm. Those of skill in the art will recognize that thepeak wavelength of the laser light used for photoablation and/orphotodetachment may have any value within this range, e.g., about 1,460nm.

As noted above, in some instances the laser used for photoablationand/or photodetachment of cells in the disclosed methods and systems mayproduce light having a bandwidth (e.g., full width at half maximum(FWHM)) centered on or near the peak wavelength that ranges from about0.0001 nm to about 10 nm, depending on peak wavelength and whether thelaser is a continuous wave laser or pulsed laser. In some instances, thebandwidth may be at least 0.0001 nm, at least 0.001 nm, at least 0.01nm, at least 0.1 nm, at least 1 nm, or at least 10 nm. In someinstances, the bandwidth may be at most 10 nm, at most 1 nm, at most 0.1nm, at most 0.01 nm, at most 0.001 nm, or at most 0.0001 nm. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the bandwidth may range from about 0.001 nm to about 1nm. Those of skill in the art will recognize that the bandwidth of thelaser light used for photoablation and/or photodetachment may have anyvalue within this range, e.g., about 0.25 nm.

As noted above, in some instances the laser used for photoablationand/or photodetachment of cells in the disclosed methods and systems mayproduce continuous wave light, and an electro-optic modulator orelectronic shutter may be used to create pulses of light of arbitrarilylong duration (e.g., ranging from tens of picoseconds to seconds). Insome instances of the disclosed methods and systems, the laser used forphotoablation and/or photodetachment of cells may be a pulsed laser andmay produce light pulses having a duration ranging from about 1femtosecond to about 100 milliseconds. In some instances, the lightpulses used for photoablation and/or photodetachment may be at least 1femtosecond, at least 1 picosecond, at least 1 nanosecond, at least 1millisecond, at least 10 milliseconds, at least 100 milliseconds, or atleast 1 second in duration. In some instances, the light pulses used forphotoablation and/or photodetachment may be at most 1 second, at most100 milliseconds, at most 10 milliseconds, at most 1 millisecond, atmost 1 nanosecond, at most 1 picosecond, or at most 1 femtosecond induration. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the light pulses used for photoablationand/or photodetachment may range from about 1 picosecond to about 1nanosecond in duration. Those of skill in the art will recognize thatthe pulse duration of the laser light used for photoablation and/orphotodetachment may have any value within this range, e.g., about 0.250nanoseconds.

As noted above, in some instances, the laser light used forphotoablation and/or photodetachment of cells in the disclosed methodsand systems may be pulsed at a pulse repetition frequency ranging fromabout 1 Hz to about 100 MHz, depending on the type of laser used. Ininstances, the pulse repetition frequency may be at least 1 Hz, at least10 Hz, at least 100 Hz, at least 1 KHz, at least 10 KHz, at least 100KHz, at least 1 MHz, at least 10 MHz, or at least 100 MHz. In someinstances, the pulse repetition frequency may be at most 100 MHz, atmost 10 MHz, at most 1 MHz, at most 100 KHz, at most 10 KHz, at most 1KHz, at most 100 Hz, at most 10 Hz, or at most 1 Hz. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the pulse repetition rate may range from about 10 Hz to about1 MHz. Those of skill in the art will recognize that the pulserepetition rate may have any value within this range, e.g., about 16.5KHz.

In some instances, the laser light irradiance (i.e., the radiant flux(power) delivered per unit area of surface, as measured, e.g., in unitsof W/cm²) may range from about 0.1 W/cm² to about 10¹⁰ W/cm², dependingon the type of laser used and the size of the focal spot at the sampleplane. In some instances, the radiant flux delivered to the samplesurface may be at least 0.1 W/cm², at least 1 W/cm², at least 10 W/cm²,at least 100 W/cm², at least 1,000 W/cm², at least 10⁴ W/cm², at least10⁵ W/cm², at least 10⁶ W/cm², at least 10⁷ W/cm², at least 10⁸ W/cm²,at least 10⁹ W/cm², or at least 10¹⁰ W/cm². In some instances, theradiant flux delivered to the sample surface may be at most at most 10¹⁰W/cm², at most 10⁹ W/cm², at most 10⁸ W/cm², at most 10⁷ W/cm², at most10⁶ W/cm², at most 10⁵ W/cm², at most 10⁴ W/cm², at most 1,000 W/cm², atmost 100 W/cm², at most 10 W/cm², at most 1 W/cm², or at most 0.1 W/cm².Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the radiant flux delivered to the samplesurface may range from about 10 W/cm² to about 1,000 W/cm². Those ofskill in the art will recognize that the radiant flux delivered to thesample surface may have any value within this range, e.g., about 0.8W/cm².

In some instances, the disclosed systems may comprise two or more lasersoperating in parallel (e.g., wherein the two laser beams are deliveredto the sample plane via the same objective, but where they comprisedifferent optical paths leading into or through the microscope orimaging module so that they can be individually targeted to differentpairs of position coordinates) such that two or more cells may beablated in parallel. In some instances, the laser light provided by asingle laser may be divided into two or more beams that are delivered tothe sample plane via the same objective, but where different opticalpaths leading into or through the microscope or imaging module are usedso that the divided beams can be individually targeted to differentpairs of position coordinates) such that two or more cells may beablated in parallel.

Translation stages: In some instances, the disclosed systems maycomprise a translation stage configured to position surfaces orcontainers, e.g., culture plate wells or other culture containers,relative to the optical axis and/or focal plane of the microscope orimaging system used to acquire images, and to position selected cellsrelative to the focal point of a laser beam delivered by means of themicroscope or imaging system objective. In some instances, the systemmay comprise a scanning mechanism, e.g. a series of programmable mirrorsor a micromirror array, configured to deliver laser light to theposition coordinates of one or more cells selected for ablation.

In some instances, the disclosed methods and systems may utilize a highprecision X-Y (or in some cases, an X-Y-Z) translation stage forre-positioning the surface or container, e.g., culture plate well orother culture container (in any of the formats described below) inrelation to the optical axis and/or focal plane of the microscope orimaging module. Suitable translation stages are commercially availablefrom a number of vendors, for example, Parker Hannifin. Precisiontranslation stage systems can comprise a combination of severalcomponents including, but not limited to, linear actuators, opticalencoders, servo and/or stepper motors, and motor controllers or driveunits. In some cases, high precision and repeatability of stage movementcan be required for the systems and methods disclosed herein in order toensure accurate positioning of individual cells targeted for ablation.Consequently, the methods and systems disclosed herein may furthercomprise specifying the precision and/or repeatability with which thetranslation stage is capable of positioning a cell in relation to theoptical axis of the microscope or imaging module, or in relation to thefocal spot of the laser light beam. In some instances, the precisionand/or repeatability of the translation stage may range from about 0.5μm to about 5 μm. In some instances, the precision and/or repeatabilityof the translation stage may be at least 0.5 μm, at least 1 μm, at least2 μm, at least 3 μm, at least 4 μm, or at least 5 μm. In some instances,the precision and/or repeatability of the translation stage may be atmost 5 μm, at most 4 μm, at most 3 μm, at most 2 μm, at most 1 um, or atmost 0.5 μm. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the precision and/orrepeatability of the translation stage may range from about 1 μm toabout 4 μm. Those of skill in the art will recognize that the precisionand/or repeatability of the translation stage may have any value withinthis range, e.g., 1.25 μm.

Containers, e.g., culture plates and other culture containers: Any of avariety of containers, e.g., culture plates or other culture containersmay be used when implementing the disclosed methods and systems.Examples include, but are not limited to, Nunc™ 6-well, 12-well,24-well, 48-well, 96-well, 384-well, and 1536-well culture plates(ThermoFisher Scientific, Waltham, Mass.), Corning® biologically-coatedculture flasks and plates (Corning, Inc. Corning, N.Y.), and theCELLSTAR® series of culture flasks, dishes, and multiwell plates fromGreiner Bio-One (Greiner Bio-One North America, Inc., Monroe, N.C.).

In some instances, one or more surfaces within the containers, e.g.,culture plate wells or other culture container used with the disclosedmethods and systems may comprise a coating designed to improve celladhesion and/or cell viability. Examples of suitable coatings include,but are not limited to, collagen, fibronectin, gelatin, laminin,poly-L-lysine, poly-D-lysine, vitronectin, and the like, or anycombination thereof.

In some instances of the disclosed methods and systems, cells may be on,or grown on, a substrate surface, e.g., the top surface of a glass,fused-silica, or polymer substrate. In some instances, the surface maycomprise partitions that divide the substrate surface into one or morediscrete regions. In some instances, the discrete regions within whichcells are cultured may be configured as one-dimensional ortwo-dimensional arrays, and may be separated from one another by meansof, e.g., a patterned hydrophobic coating or thin metal layer. In otheraspects, the discrete regions may comprise indents in the substratesurface. In still other aspects, the discrete regions may be separatedfrom each other by means of a well-forming component such that thesubstrate forms the bottom of a microwell plate (or microplate), andeach individual discrete region forms the bottom of one well in themicrowell plate. In one aspect of the present disclosure, thewell-forming component separates the top surface of the substrate into96 separate wells. In another aspect, the well-forming componentseparates the top surface of the substrate into 384 wells. In yetanother aspect, the well-forming component separates the top surface ofthe substrate into 1,536 wells. In all of these aspects, the substrate,whether configured in a planar array, indented array, or microwell plateformat, may comprise a disposable or consumable device or cartridge thatinterfaces with other optical and mechanical components of the disclosedsystems.

The methods and systems disclosed herein may further comprise specifyingthe number of discrete regions or wells into which the substrate surfaceis divided, irrespective of how separation is maintained betweendiscrete regions or wells. Having larger numbers of discrete regions orwells on a substrate may be advantageous in terms of increasing theprocessing throughput of the disclosed methods. In one aspect of thepresent disclosure, the number of discrete regions or wells persubstrate may range from about 10 to about 1,600. In other aspects, thenumber of discrete regions or wells may be at least 10, at least 20, atleast 50, at least 100, at least 200, at least 300, at least 400, atleast 500, at least 750, at least 1,000, at least 1,250, at least 1,500,or at least 1,600. In yet other aspects of the disclosed methods andsystems, the number of discrete regions or wells may be at most 1,600,at most 1,500, at most 1,000, at most 750, at most 500, at most 400, atmost 300, at most 200, at most 100, at most 50, at most 20, or at most10. In a preferred aspect, the number of discrete regions or wells is96. In another preferred aspect, the number of discrete regions or wellsis 384. In yet another preferred aspect, the number of discrete regionsor wells is 1,536. Those of skill in the art will appreciate that thenumber of discrete regions or wells may fall within any range bounded byany of these values (e.g. from about 12 to about 1,400), and may haveany value within this range, e.g., 25 discrete regions or wells.

The methods and systems disclosed herein may also comprise specifyingthe surface area of the discrete regions or wells into which thesubstrate surface is divided, irrespective of how separation ismaintained between discrete regions or wells. Having discrete regions orwells of larger area may facilitate ease-of-access and manipulation ofthe cultured cells in some cases, whereas having discrete regions orwells of smaller area may be advantageous in terms of reducing culturemedium volume requirements and/or increasing the processing throughputof the disclosed methods. In one aspect of the present disclosure, thesurface area of the discrete regions or wells is between 1 mm² and 100mm². In other aspects, the area of the discrete regions or wells is atleast 1 mm², at least 2.5 mm², at least 5 mm², at least 10 mm², at least20 mm², at least 30 mm², at least 40 mm², at least 50 mm², at least 75mm², or at least 100 mm². In yet other aspects of the disclosed methodsand systems, the area of the discrete regions or wells is at most 100mm², at most 75 mm², at most 50 mm², at most 40 mm², at most 30 mm², atmost 20 mm², at most 10 mm², at most 5 mm², at most 2.5 mm², or at most1 mm². In a preferred aspect, the area of discrete regions or wells isabout 35 mm². In another preferred aspect, the area of the discreteregions or wells is about 8.6 mm². Those of skill in the art willappreciate that the area of the discrete regions or wells may fallwithin any range bounded by any of these values (e.g. from about 2 mm²to about 95 mm²) and may have any value within this range, e.g., 64 mm².

Environmental control chamber or module: In some instances, the systemsof the present disclosure may comprise an environmental control chamberor module for maintaining the cells to be imaged and selected forretention or destruction at a specified temperature, humidity, O₂concentration, CO₂ concentration, N₂ concentration, etc., or anycombination thereof. In some instances, the environmental chamber mayencompass all or a portion of the remaining system components. In someinstances, the environmental chamber may be configured to make contactwith or encompass only the cell culture containers being processed,e.g., it maybe sized and adapted to be mounted on the translation stage.

Examples of environmental control chambers that may be suitable forimplementation of the disclosed methods and systems include, but are notlimited to, the enclosures available from Okolab (San Bruno, Calif.),Olympus (Center Valley, Pa.), Zeiss (Thornwood, N.Y.), and others.

Fluid-handling robotics: As noted above, in some instances the systemsdisclosed herein may further comprise an automated, programmablefluid-dispensing (or liquid-dispensing) system for use in depositingcells from a dilute suspension into culture plate wells or other culturecontainers. Suitable automated, programmable fluid-dispensing systemsare commercially available from a number of vendors, e.g., BeckmanCoulter Life Sciences (Indianapolis, Ind.), Perkin Elmer (Waltham,Mass.), Tecan (Baldwin Park, Calif.), Agilent—Velocity 11 (Menlo Park,Calif.), and many others. In some aspects of the disclosed methods andsystems, the fluid-dispensing system may comprise a multichanneldispense head, e.g. a 4 channel, 8 channel, 16 channel, 96 channel, or384 channel dispense head, for simultaneous delivery of programmablevolumes of a cell suspension, culture medium, or other liquid (e.g.,volumes ranging from about 1 microliter to several milliliters) tomultiple culture plate wells, locations on a substrate surface, etc.

Plate handling robotics: In some instances, the systems disclosed hereinmay further comprise a culture plate-handling (or culturecontainer-handling) robotic system for automated replacement andpositioning of culture plates or containers (in any of the formatsdescribed above) in relation to the fluid-dispensing system and/or inrelation to the translation stage and/or optical axis of the microscopeor imaging module. Suitable automated, programmable plate-handlingrobotic systems are commercially available from a number of vendors,including Beckman Coulter Life Sciences (Indianapolis, Ind.), PerkinElmer (Waltham, Mass.), Tecan (Baldwin Park Calif.), Agilent—Velocity 11(Menlo Park, Calif.), and many others. In some aspects of the methodsand systems disclosed herein, the automated plate-handling roboticsystem may be configured to move containers, e.g., culture plates orculture containers back and forth between the disclosed laser ablationsystems and longer-term cell culture incubators.

Processors, controllers, or computers: In some instances, the disclosedsystems may comprise one or more processors, controllers, or computersthat are configured to execute programmable, software-encodedinstructions for: (i) setting and maintaining the environmentalparameters within an environmental control chamber (e.g., temperature,humidity, O₂ concentration, CO₂ concentration, etc.) to optimize and/ormaintain cell viability during imaging, (ii) controlling illuminationlight settings (e.g., intensity and wavelength) and image acquisition(e.g., exposure time, exposure frequency, number of images acquired,etc.), (iii) controlling image pre-processing (e.g., correction of imagecontrast and brightness, correction for non-uniform illumination,correction for an optical aberration, removal of noise, etc., or anycombination thereof) and/or image processing (identification of objects(e.g., cells or sub-cellular structures) within each of the images in aseries of one or more images, segmentation of each image to isolate theidentified objects, tiling of segmented images to create compositeimages, performing feature extraction (e.g., identification and/orquantitation of object properties such as observable cellular phenotypictraits), determining the position coordinates for one or more selectedcells, determining a confidence level for the destruction of theselected cells from one or more images acquired after performing theablation step etc., or any combination thereof), (iv) controlling thelaser targeting system for ablating unwanted cells (e.g., reading theposition coordinates of the selected cells and re-positioning thetranslation stage such that the selected cells are sequentiallypositioned within the focal spot of the laser beam; controlling theintensity and/or duration of the laser light to which the selected cellsare exposed), or any combination of these steps.

In some instances, the one or more processors, controllers, or computersof the disclosed systems may be further configured to executeprogrammable, software-encoded instruction for controlling thedeposition of cells from a dilute cell suspension into containers, e.g.,culture plate wells or other culture containers, using a roboticfluid-dispensing system.

In some instances, the one or more processors, controllers, or computersof the disclosed systems may be further configured to executeprogrammable, software-encoded instruction for controlling aplate-handling robotic system that moves surfaces or containers, e.g.,culture plates or other culture containers, back and forth between thelaser ablation system and long-term cell culture incubators.

In some instances, the one or more processors, controllers, or computersof the disclosed systems may comprise a network interface (andassociated software) for transferring surface or container, e.g.,culture plate tracking data, image data, cell identification andablation data, and/or other experimental data from the laser ablationsystem to a laboratory information management system (LIMS).

In some instances, the one or more processors of the disclosed systemsmay comprise a hardware processor such as a central processing unit(CPU), a graphic processing unit (GPU), a general-purpose processingunit, or computing platform. The one or more processors may be comprisedof any of a variety of suitable integrated circuits (e.g., applicationspecific integrated circuits (ASICs) designed specifically forimplementing the disclosed image processing-based methods, orfield-programmable gate arrays (FPGAs) to accelerate compute time, etc.,and/or to facilitate deployment), microprocessors, emergingnext-generation microprocessor designs (e.g., memristor-basedprocessors), logic devices and the like. Although the disclosure isdescribed with reference to a processor, other types of integratedcircuits and logic devices may also be applicable. The processor mayhave any suitable data operation capability. For example, the processormay perform 512 bit, 256 bit, 128 bit, 64 bit, 32 bit, or 16 bit dataoperations. The one or more processors may be single core or multi coreprocessors, or a plurality of processors configured for parallelprocessing.

The one or more processors or computers used to implement the disclosedmethods may be part of a larger computer system and/or may beoperatively coupled to a computer network (or a “network”) with the aidof a communication interface to facilitate transmission of and sharingof data and surface or container, e.g., culture plate processingresults. The network may be a local area network, an intranet and/orextranet, an intranet and/or extranet that is in communication with theInternet, or the Internet. The network in some cases is atelecommunication and/or data network. The network may include one ormore computer servers, which in some cases enables distributedcomputing, such as cloud computing. The network, in some cases with theaid of the computer system, may implement a peer-to-peer network, whichmay enable devices coupled to the computer system to behave as a clientor a server.

The computer system may also include memory or memory locations (e.g.,random-access memory, read-only memory, flash memory, Intel® Optane™technology), electronic storage units (e.g., hard disks), communicationinterfaces (e.g., network adapters) for communicating with one or moreother systems, and peripheral devices, such as cache, other memory, datastorage and/or electronic display adapters. The memory, storage units,interfaces and peripheral devices may be in communication with the oneor more processors, e.g., a CPU, through a communication bus, e.g., asis found on a motherboard. The storage unit(s) may be data storageunit(s) (or data repositories) for storing data.

The one or more processors, e.g., a CPU, execute a sequence ofmachine-readable instructions, which are embodied in a program (or“software”). The instructions are stored in a memory location. Theinstructions are directed to the CPU, which subsequently program orotherwise configure the CPU to implement the methods of the presentdisclosure. Examples of operations performed by the CPU include fetch,decode, execute, and write back. The CPU may be part of a circuit, suchas an integrated circuit. One or more other components of the system maybe included in the circuit. In some cases, the circuit is an applicationspecific integrated circuit (ASIC).

In some instances, a computer system of the present disclosure maycomprise a storage unit that stores files, such as drivers, librariesand saved programs. The storage unit may store user data, e.g.,user-specified preferences and user-specified programs. The computersystem in some cases may include one or more additional data storageunits that are external to the computer system, such as located on aremote server that is in communication with the computer system throughan intranet or the Internet.

Software: Some aspects of the methods and systems provided herein, suchas the disclosed methods for selecting and ablating cells in a cultureplate well, are implemented by way of machine-executable code(processor-executable code) stored in an electronic storage location ofthe computer system, such as, for example, in the memory or electronicstorage unit. The machine-executable or machine-readable code isprovided in the form of software. During use, the code is executed bythe one or more processors. In some cases, the code is retrieved fromthe storage unit and stored in the memory for ready access by the one ormore processors. In some situations, the electronic storage unit isprecluded, and machine-executable instructions are stored in memory. Thecode may be pre-compiled and configured for use with a machine havingone or more processors adapted to execute the code or may be compiled atrun time. The code may be supplied in a programming language that isselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Various aspects of the disclosed method and systems may be thought of as“products” or “articles of manufacture”, e.g., “computer program orsoftware products”, typically in the form of machine (or processor)executable code and/or associated data that is stored in a type ofmachine readable medium, where the executable code comprises a pluralityof instructions for controlling a computer or computer system inperforming one or more of the methods disclosed herein.Machine-executable code may be stored in an optical storage unitcomprising an optically readable medium such as an optical disc, CD-ROM,DVD, or Blu-Ray disc. Machine-executable code may be stored in anelectronic storage unit, such as memory (e.g., read-only memory,random-access memory, flash memory) or on a hard disk. “Storage” typemedia include any or all of the tangible memory of the computers,processors or the like, or associated modules thereof, such as varioussemiconductor memory chips, optical drives, tape drives, disk drives andthe like, which may provide non-transitory storage at any time for thesoftware that encodes the methods and algorithms disclosed herein.

All or a portion of the software code may at times be communicated viathe Internet or various other telecommunication networks. Suchcommunications, for example, enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, other types of media that are used to convey the softwareencoded instructions include optical, electrical and electromagneticwaves, such as those used across physical interfaces between localdevices, through wired and optical landline networks, and over variousatmospheric links. The physical elements that carry such waves, such aswired or wireless links, optical links, or the like, are also consideredmedia that convey the software encoded instructions for performing themethods disclosed herein. As used herein, unless restricted tonon-transitory, tangible “storage” media, terms such as computer ormachine “readable medium” refer to any medium that participates inproviding instructions to a processor for execution.

The computer system typically includes, or may be in communication with,an electronic display for providing, for example, images captured by amachine vision system. The display is typically also capable ofproviding a user interface (UI). Examples of UI's include but are notlimited to graphical user interfaces (GUIs), web-based user interfaces,and the like.

FIG. 3 provides a block diagram for system control software used tocontrol a laser photoablation system according to one aspect of thepresent disclosure. In some instances, the control software may comprisemachine-readable or machine-executable instructions for communicatingwith and/or controlling: (i) an image acquisition module, (ii) an imageprocessing module, (iii) an ablation control module (e.g., a celltargeting and laser exposure control module), (iv) an environmentcontrol module, and/or (v) a LIMS interface, or any combination of thesesteps. In some instances the system control software may furthercomprise software for interfacing the laser ablation systems of thepresent disclosure with: (vi) a fluid-handling system used to dispensecells onto a surface or into a container, e.g., culture plate wells orother culture containers, and/or (vii) a robotic plate-handling systemfor moving surfaces or containers, e.g., culture plates or other culturecontainers, back and forth between the fluid-handling system, the laserablation system, and/or a long-term cell culture incubator.

In some instances, the system control software and all component modulesthereof may be executed by a single processor or computer. In someinstances, the system control software and one or more of the componentmodules may be performed on different processors or computers. In someinstances, all or a portion of the system control software and/orcomponent modules thereof may be performed by a computer network and/orcloud-based computing system.

System performance specifications: In general, the systems disclosedherein will be configured to perform the disclosed methods for cellidentification, selection, and ablation with performance specificationsfor photoablation rate, percentage of cells ablated, efficiency forrendering ablated cells as non-viable, processing throughput, andpercentage of processed surfaces or containers, e.g., culture platewells or culture containers, containing a single viable cell followingthe photoablation step as described under the methods section above.

Applications: The methods and systems disclosed herein are generallyapplicable to the preparation of clonal populations of cells. Examplesof specific applications to which the disclosed methods and systems maybe applied include, but are not limited to, destruction ofundifferentiated stem cells in an in vitro differentiated stem cellculture, destruction of differentiated stem cells in an in vitroundifferentiated stem cell culture, isolation of single stem cells andpreparation of clonal stem cell colonies, isolation of single cells(e.g., cells expressing a successful CRISPR editing parameter) andpreparation of clonal cell colonies therefrom, preparation of clonalpopulations of plant cells, and the like.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1—Prototype Laser Ablation System

A prototype imaging-based cell targeting and laser ablation system wasconstructed that comprised: (i) a microscope (Zeiss (Jena, Germany) AxioObserver 7) for viewing cells in culture dishes and plates, (ii) a Zeissmicroscope focusing system for keeping cells in focus, (iii) a laser(Hamilton Thorne (Beverly, Mass.) Stiletto laser) and objective(Hamilton Thorne (Beverly, Mass.) 20× Objective) that are capable ofworking in tandem to focus light on a specific location in the culturecontainer, (iv) a camera system (Lumenera (Ottawa, Canada) Infinity3camera) used to record the location, presence, and absence of cells inthe culture container, (v) custom software that performed image captureand ablation control (which may optionally be updated for integrationwith a software control structure that tracks the process (to keep trackof the clonal production if the ablation system is part of a largerworkflow), and (vi) a custom environmental control system that kept thecontainer holding the cells to be ablated under a specified set ofculturing conditions (5% CO₂ and 37C; the gas mixture was bubbledthrough water to provide constant humidity).

FIG. 4 provides a CAD model (isometric view) and FIG. 5 provides a CADmodel (side view) of the inverted microscope used to assemble theprototype laser ablation system. As can be seen in the model views, aculture flask was positioned on the microscope stage and viewed frombelow using the inverted microscope optics. A light beam from a laser(not shown) was directed into the rear of the microscope base anddelivered to the sample plane via the overhead optical path.

FIG. 6 provides a CAD model of the microscope of FIGS. 4 and 5 alongwith optical elements used to couple laser light from a diode laseroperating at approximately 1,440-1,450 nm at a pulse rate ofapproximately 16 pulses per millisecond into the microscope, and ahousing (removed in this view) used to control the imaging environment.

HEK293 and HT1080 cells were plated in culture plates and grown in acomplete serum-free, low-protein 293 SFMII growth medium (Thermo-FisherScientific, Waltham, Mass.) supplemented with 4 mM L-glutamine at 37Cand 5% CO₂. Custom software was used to program the targetingcoordinates of cells for ablation. The culture plates were monitoredover the course of several days to ensure that the retained cellssurvived.

FIGS. 7A-B show non-limiting examples of data that illustrate the use ofthe prototype laser ablation system to remove HEK293 cells and createpatterns in a confluent cell culture. FIG. 7A: a pattern of cells wasdestroyed by laser photoablation according to a specified set ofposition coordinates (box). FIG. 7B: a subsequent photoablation patternperformed on the same cell culture according to an updated set ofposition coordinates (box).

FIGS. 8A-B show non-limiting examples of bright-field image data thatillustrate the use of a prototype laser ablation system to destroysingle HEK293 cells. FIG. 8A: image of a cell culture plate showingthree targeted single cells as indicated in the boxes. FIG. 8B: image ofthe same cell culture plate following the destruction of the targetedcells by laser photoablation.

Example 2—Laser Power Study

The software for a commercially-available Hamilton Thorne Stiletto®(Hamilton Thorne, Beverly, Mass.) laser system allows the user tocontrol laser power (% of maximum; 300 mW peak power), laser pulselength (100-3000 μs), and pulse frequency (1-200 Hz). We have modifiedthe software that integrates and controls the components of the lasersystem, e.g., a laser, translation stage, and camera, to enableindependent application development. In this example, we have modifiedthe system integration software to enable operation in a continuous modewhere laser power (%) can be changed, and to enable operation in singlelaser pulse mode where both laser power (%) and pulse length (μs) can becontrolled. A study was designed to determine optimal laser power levelsfor ablating cells from a given area using a continuous raster patternwithout killing neighboring cells, as well as to determine the optimalpower and pulse length requirements for killing individual cells usingsingle laser pulses. These measurements were made in confluent sheets ofHEK293 cells, and cell death was determined by propidium iodide (PI)fluorescence, a marker of cell membrane integrity where dead cellsfluoresce red. For raster mode measurements, a 10 μm×10 μm square wasablated at 10%-100% laser power with 24 replicates per condition. Forsingle pulse mode, a point was ablated using 100-300 μs pulse lengthsand 10-100% laser power with 3 replicates per condition.

FIG. 9 provides a non-limiting example of laser photoablation data wherecells were selectively ablated using a continuous raster pattern ofexposure to laser light. Using 20% laser power was sufficient to killcells in a small area. Increasing laser power expands the killing area,while using 60% laser power or higher resulted in detachment of cells.

FIG. 10 provides a non-limiting example of laser photoablation datawhere cells were selectively ablated using exposure to single pulses oflaser light. Single pulse point ablation did not produce an unambiguouspattern of cell killing. Use of 100% laser power resulted in the killingof clusters of cells at several longer pulse lengths (300 μs, 280 μs,260 μs), but there was considerable variability in the percentage ofcells killed.

These results demonstrate that the use of continuous rastering of laserlight at a laser power lower than 20% was insufficient to kill cells,while the use of laser power levels of greater than 40% led toincreasingly large areas of cell death for a 10 μm×10 μm raster area.The efficacy of single pulse laser irradiation proved to be highlyvariable in this study, with long (260+μs), high power (70%+) pulsescapable of killing multiple cells per area. However, the data for singlepulse irradiation appears to be considerably noisier than that for areaablation.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in any combination in practicing the invention.It is intended that the following claims define the scope of theinvention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A method comprising: a) selecting a first cell onone or more surfaces or in one or more containers, wherein the selectingis not based on whether the first cell comprises an exogenous label oran expressed reporter; and b) photoablating at least 80% of the cells oneach of the one or more surfaces or in each of the one or morecontainers, wherein at least 90% of the one or more surfaces orcontainers contain only one viable cell after the photoablation isperformed, wherein the first cell selected in (a) is not photoabalated.2. The method of claim 1, wherein at least 95% of the one or moresurfaces or containers contain only one viable cell after thephotoablation is performed.
 3. The method of claim 1, wherein at least98% of the one or more surfaces or containers contain only one viablecell after the photoablation is performed.
 4. The method of claim 1,wherein the one or more surfaces or containers comprise at least 5, 10,20, 30, 40, 50, 60, 70, 80, 90, or 100 surfaces or containers.
 5. Themethod of claim 1, wherein the first cell is selected using an imagingtechnique.
 6. The method of claim 5, wherein the imaging techniquecomprises bright-field imaging, dark-field imaging, phase contrastimaging or any combination thereof.
 7. The method of claim 1, whereinthe selecting is based on position of the first cell on one or moresurfaces or in one or more containers.
 8. The method of claim 7, whereinthe selecting is based on proximity of the first cell to a center of theone or more surfaces or containers.
 9. The method of claim 1, whereinthe selecting is based on size of the first cell.
 10. The method ofclaim 1, wherein the selecting is based on a morphology of the firstcell.
 11. The method of claim 1, wherein the selecting is based on aphenotype of the first cell.
 12. The method of claim 1, wherein theselecting is based on a development stage of the first cell.
 13. Themethod of claim 1, wherein cells are photoablated at a rate in the rangeof 10 cells per minute to 200 cells per minute.
 14. The method of claim1, wherein cells are photoablated using light in the wavelength range of220 nm to 1500 nm.
 15. The method of claim 14, wherein cells arephotoablated using light in the wavelength range of 1440 nm to 1450 nm.16. The method of claim 1, further comprising growing a clonalpopulation of the first cell after the photoablation is performed. 17.The method of claim 16, further comprising performing an assay on theclonal population of the first cell.
 18. The method of claim 16, furthercomprising photodetaching one or more cells of the clonal population ofthe first cell from the one or more surfaces or one or more containersthereby obtaining one or more photodetached cells.
 19. The method ofclaim 18, further comprising performing an assay on the one or morephotodetached cells.
 20. The method of claim 18, further comprisinggrowing the one or more photodetached cells.